Mechanisms of Apoptosis and Cytotoxicity

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Mechanisms of Apoptosis and Cytotoxicity
Yufang Shi, Ph.D. Shiyu_at_umdnj.edu
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1. Introduction2. History3. Detection of
Apoptotic Cells4. Apoptosis in
Invertebrates5. Mechanisms of
Apoptosis Bcl-2 family Caspases Apaf I
APs TNF family Granzymes/Cytotoxicity p
53 pathway 6. Cytotoxicity7. Autophagy and
autophagic cell death
Mechanisms of Apoptosis and Cytotoxicity
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Introduction
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In our body, millions of cells die every minute
Dead skin cells are shed off 30,000 - 40,000
cells every minute 4-10 x 107 of 1014 cells in
the human body undergo apoptosis daily
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Characteristics of Apoptosis and Necrosis

• Apoptosis
• Fragmentation of genomic
• DNA into a 200 bp ladder
• Condensation of chromatin
• and formation of apoptotic body
• Initiated by a signal transduction
• process
• An active process requires
• macromolecule synthesis
• 5. Does not cause inflammation

• Necrosis
• No fragmentation of genomic
• DNA into a 200 bp ladder
• No condensation of chromatin
• and no formation of apoptotic body
• Initiated by direct cell damage
• mostly physical
• An passive process does not require
• macromolecule synthesis
• 5. Cause inflammation

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A Schematic Representation of Apoptosis Pathways
DIABLO
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Publications in Apoptosis Research
162,822 papers to date
15,000 publications/year since 2000, corresponds
to 1.8 of all papers in life sciences. 40 of
all publications receive no citations in the
subsequent literature, and 2/3 of the rest
receive less than two citations.
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Significance of Apoptosis
Anti-CD3 Anti-CD28
5 x 106 T Cells
1017
65 days
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• The Role of Apoptosis in the Immune System
• Selection of the T and B cell repertoire
• Regulation of peripheral lymphocyte homeostasis
• Cytototic killing of target cells
• Corticosteroid/Stress-induced immune suppression
• Cytokine-withdrawal-induced apoptosis
• Cytokine-activation-induced apoptosis

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Activation-Induced Cell Death (AICD)

• In T cell hybridomas
• In immature T cells as a mechanism of negative
  selection
• In activated T cells for the maintenance of
  peripheral
• lymphocyte homeostasis
• In B cell lymphomas and mature B cells
• AICD in T cell hybridomas and mature T cells has
  been
• shown to be mediated by the interaction between
  Fas and FasL

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Outcomes of TCR Ligation
Immature T cells
Mature T cells Proliferate
Activated T cell blasts AICD
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Apoptosis and Cancer Development of Cancer
Increase in cell viability and decrease in
apoptosis Protooncogenes regulate
apoptosis Cancer therapy Induce apoptosis
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History
Vaux DL. Apoptosis timeline. Cell Death Differ.
2002, 9349-54.
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History The word apoptosis was introduced by
Kerr, Wyllie and Currie (1972, Br.J.Cancer
26239-257) to describe a form of cell death
distinct from necrosis. Apo Greek from,
separation from Ptosis Greek dropping, a
falling down It means falling of parts in
ancient Greek. In the book Mochlicon (460-370
BC) a p o p t w s i e s, dislocations of the
bones, structural changes (bone erosion) related
to tissue and cell death! Galen (129-201 AD)
extended its medical meaning to "dropping of the
scabs" and given the role of apoptosis in wound
healing and inflammation. Marcus Aurelius
(Galen was his physician) in political and
social contexts, apoptosis becomes a synonymous
of failure, ruin, decline and decadence
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Milestones in Apoptosis Research
(1) Virchow-1855 All diseases are due to active
or passive disturbances in cells Changes in
morphology correlate with function      ((The
CELL THEORY was introduced in 1839 by Schleiden
Schwann) Schmauss-1895 Hyperchromatosis,
Karyorrhexis Karyolysis Glucksmann-1930-1951 Ce
ll death is a normal accompaniment of vertebrate
ontogeny The feature of physiological cell
death -Chromatopycnosis and hyperchromatosis -S
hrinkage of nucleus and cell due to fluid
loss -No changes in mitochondria -Degenerating
cells are often phagocytoses Cells exposed to
injurious agents have different morphology
Necrosis -Swelling of mitochondria and cell
itself -Karyolysis Glucksmann A. Cell death in
normal vertebrate ontogeny Biol Rev 1951 26-
59-86
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Schmauss, 1895
Pychotic
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Very First Electromicrograph of Chromatin
Condensation
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Milestones in Apoptosis Research (2)
Lockshin-1964 Cell death during invertebrate
development is programmed The term programmed
cell death was proposed Tada-1966 Inhibitors of
RNA and Protein synthesis blocked cell death
during amphibian metamorphosis -An active
process requiring protein synthesis Saunders-1966
Death in embryogenesis -Suicide, not
assassination
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Milestones in Apoptosis Research (3)
Kerr, Wyllie and Currie-1972 Apoptosis -A basic
biological phenomenon with wide-ranging
implications in tissue kinetics -Controlled
cell deletion which occurs under physiological
conditions in adults -Morphologically identical
to developmental cell death as described by
Glucksmann, also called cell shrinkage, and
distinct from accidental cell death or
coagulative necrosis -Active, controlled process,
the deregulation of which could lead to many
diseases including cancer
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Kerr, Wyllie, and Currie, 1972
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Milestones in Apoptosis Research (4)
Wyllie 1980 Biochemical changes which accompany
apoptotic morphology -DNA is degraded into a
nucleosome ladder DNA fragmentation probably
due to activation of Ca2/Mg2-dependent
endogenous endonuclease as first described by
Hewish and Burgoyne in 1970 Internucleosomal
DNA fragmentation first described in cells
undergoing growth factor deprivation
(Williamson, 1970) and in irradiated
lymphocytes (Skalka, et al., 1976) -Requires
synthesis of death genes.
Andrew Wyllie
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Milestones in Apoptosis Research (5)

• Bcl-2 was found to be anti-apoptotic
• 1991. ced (cell death abnormal) genes were
  identified in Caenorhabditis elegans
• Fas/APO-1/CD95 identified.
• A role of p53 in regulation of apoptosis was
  proposed.
• Fas ligand identified.
• ICE was found to be the mammalian homolog of
  ced-3.
• IAPs (inhibitors of apoptosis) were identified
• 1994. Mitochondria were proposed to be a primary
  target during apoptosis.
• 1996. The term caspase was coined to represent a
  group of proteases
• that are activated during apoptosis.
• Leakage of cytochrome c from mitochondria during
  apoptosis was recognized.
• 1997. Apafs (apoptosis-associated factors)
  identified.

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Detection of Apoptotic Cells
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Morphology
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Electromicroscopy
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Histology
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Genomic DNA Fragmentation
DNA Content Analysis
Membrane Permeability
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From Blankenberg, Stanford University
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Annexin V Staining
PI
Annexin
Translocase and floppase inactivated,
scramblase activate
From Blankenberg, Stanford University
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Terminal Deoxynucleotidyl Transferase-mediated
dUTP Nick End Labeling (TUNEL)
Caspases MTT PARP cleavage
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Apoptosis in Invertebrates
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The C. elegans System
Sydney Brenner H. Robert Horvitz John E.
Sulston
Caenorhabditis elegans
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The C. elegans System Cell Death Genes
ced (cell death abnormal) mutant was generated in
1983
Happy cells Commitment to die
Phagocytosis Degradation
and cell suicide of dying cell
of cell
Ced-3 caspases Ced-9 Bcl-2 EGL-1Bid/Bad Ced-4A
paf-1
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The C. elegans System Phagocytosis
ced-4 activates ced-3 to kill the 130
cells ced-9 inhibits ced-3
From Zheng Zhou www.bcm.edu/biochem/
fac/zhou/zhou.html
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Homologues in the Apoptosis Pathways
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Apoptosis system in Drosophila and Mammals
Dark mutation lead to resistance to
Radiation. Dmp53 and Rpr is induced by
radiation.
1

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J Immunol Methods 2002 Jul 1265(1-2)21-38
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Mechanisms of Apoptosis
Bcl-2 family Caspases Apaf IAPs TNF
family Granzymes/Cytotoxicity
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Key intracellular regulators of apoptosis
(1) Bcl-2 proteins (20 members) B cell
leukemia/lymphoma 2 Translocation between
Chromosome 14 and 18 results in the promoter of
IgH control bcl-2 expression 20 members.
homologs of ced-9, some are anti-apoptotic (Bcl-2
, bcl-XL), inhibiting apoptosis by
heterodimerization with pro-apoptotic members, by
blocking Cytochrome C release or by binding to
Apaf-1. Others are pro-apoptotic (Bax, bak, Bad).
Caspases cysteine-dependent
asparate-specific proteases homologs of C.elegan
ced-3 14 identified caspase active site
QACxG Apaf-1 apoptosis protease activating
factor 1 homolog of ced-4, necessary for caspase
(caspase-9,2) activation form apoptosome with
cytochrome c, Smac/DIABLO, and caspase-9.
Adams and Cory, 2007. Oncogene
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Key intracellular regulators of apoptosis (2)
IAPs Inhibitor of apoptosis (inhibit effector
caspase activity and promote degradation). 8 in
human cells. Also found in viruses Human
c-IAP-1, c-IAP-2, XIAP, Survivin, ILP-2, ML-IAP,
NAIP and Bruce Two domain types Bir domain and
RING-Zn Finger Smac/Diablo inbibit IAPs
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The Bcl-2 Family Proteins
BH3-only Bcl2 proteins are proapoptotic Bid,
Bim and EGL-1
Adams and Cory, 2007. Oncogene
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BH Domains

• Bcl-2 homology (BH) domains BH1, BH2, BH3 and
  BH4.
• Pro-apoptotic members require the BH3 domain for
  their function.
• The BH domains do not have any enzymatic
  activity, instead function by dimerization.
• BH1 and BH2 in death antagonists, allow
  heterodimerization with Bax to repress apoptosis.
• BH3 in death agonists (eg. Bax, Bak) allows
  heterodimerization with Bcl-XL and Bcl-2 to
  promote apoptosis.
• BH4 conserved in apoptosis antagonist members
  (eg. Bcl-XL), allows interaction with death
  regulatory proteins such as Raf-1, Bad, and
  perhaps Ced-4.
• Bid only one BH3. Caspase 8 cleavage of Bid
  results in translocation of cleaved Bid to the
  mitochondria inducing Cyt. C release. Thus, Bid
  may function to link the death receptor pathway
  with the mitochondrial cell death pathway in
  order to amplify caspase activation.

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General Function of Bcl-2 Family Proteins
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Caspases (1)
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Caspases (2)
Long prodomain 1,2,4,5,8,9,10,11,12 interact
with DED (8,10) or CARD (1,2,4,5,9,11,12,13) Caspa
se 1,4,5 are activated by Toll receptors/TLRs Sub
strates Caspase 8LETD Caspase 3
DEVD Caspase-9 LEHD Cytokine processing Lamin Ge
lsolin FAK(focal adhesion kinase) PAK2
(P21-activated kinases) PARP ICAD
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Apaf-1
Cyt. C interacts Apaf-1, dATP/ATP and pro-caspase
9 to form a complex called the "apoptosome"
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Inhibitors of Apoptosis (IAPs)

• Originally identified in 1993 from baculoviruses,
  IAPs also referred to as baculoviral IAP repeat
  (BIR)-containing proteins (BIRPS). BIRPS were
  found to suppress the host cell apoptotic
  response to viral infection.
• Baculovirus Cp-IAP, Op-IAP
• Humans, cellular-IAP-1 (c-IAP-1), c-IAP-2,
  X-chromosome-linked IAP (XIAP), Survivin,
  IAP-like protein-2 (ILP-2), melanoma IAP
  (ML-IAP), neuronal apoptosis inhibitory protein
  (NAIP) and BIR repeat-containing
  ubiquitin-conjugating enzyme (BRUCE).
• Overexpression inhibits apoptosis
• IAPs do not bind to nor inhibit caspase 8 IAPs
  bind to and inhibit caspase 3,7,9
• e.g. survivin expressed during development, not
  in terminally differentiated cells. C-IAP1,
  c-IAP2, XIAP, Survivin expressed in most tumors,
  correlates to prognosis. Small molecule
  inhibitors induce apoptosis and are now being
  developed for chemo.

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IAP Domains

• Two motifs 1) the Baculoviral Inhibitor of
  apoptosis Repeat (BIR) domain
• 2) the COOH terminus RING (really interesting
  new gene) Zn finger.
• Each IAP contains one to three BIR domains, but
  only one is necessary for caspase inhibition.
• RING Zn finger is involved in DNA and protein
  interactions. Has ubiquitin protein ligase
  activity that promotes caspase 3 degradation,
  thereby enhancing the anti-apoptotic activity of
  IAPs.
• IAPs inhibit caspase 9 function by binding to
  inactive pro-caspase 9 and obstructing
  pro- caspase 9 processing and activation
• During AICD, XIAP and c-IAP-1
• are ubiquitinated and degraded in
• proteasomes and it is an essential step.
• Smac only interact with Bir2
• (caspase3,7) and Bir3 (caspase 9),
• but not Bir1

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Viral Caspase Inhibitors
CrmA (Cytokine response modifier A) a viral gene
product from the Cowpox virus that is a natural
caspase inhibitor CrmA resembles members of the
serine protease inhibitor (serpin) family and is
cleaved by the caspases it inhibits, caspase 1
and caspase 8. CrmA irreversibly binds to
caspase 8, thus inhibiting its activity P35 a
baculoviral gene product that inhibits several
caspases including caspase 1, caspase 3, caspase
6, caspase 7, caspase 8, and caspase 10. P35 is
hydrolyzed by its caspase targets and remains
tightly bound to its active site. To date, no
mammalian homolog of p35 have been found.
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The TNF Superfamily and Receptors
Share 20-30 homology Soluble forms can be
generated by proteolysis Ligands 19, type
II Receptors 29, type I

• Function
• Control the immune and inflammatory responses
• Costimulation
• Development of secondary lymphoid organs
• Cytotoxic effector and homeostasis of the
  lymphoid system
• Development and differentiation of epithelial
  structures
• Bone homeostasis
• Mammary gland differentiation.
• Formation of hair follicles

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TNF Receptor Superfamily (TNFRSF)
Light
TR6
OPG
RANKL
TRAIL
FasL
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Fas-mediated death signaling
Receptor trimerization and DISC formation
Ligand Binding
Ligand Binding
Activation of procaspase-8 and downstream caspases
Fas
Cleavage of cellular death substrates
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Fas-Mediated Apoptosis
Ligand Binding
Ligand Binding
Receptor trimerization and DISC formation
Receptor trimerization and DIS C formation
Activation of procaspase-8 and downstream caspases
Activation of procaspase-8 and downstream caspases
Cleavage of cellular death substrates
Cleavage of cellular death substrates
DISC death-inducing signaling complexes
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Type I and Type II Cells
Type II Do not require F-actin Bid
activation Has less Fas Sensitive to soluble FasL
Do not form Disc
Type I Require F-actin for internalization No
Bid activation Has more Fas Not sensitive to
soluble FasL Form Disc
No Caspase-8 activation or less Fas leads to
NFkBThus tumors down regulate Fas for ssurvival
(This is not abserved in TRAILRs)
From Daivs and Reuda, 2002
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TNF-Mediated Apoptosis
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Cytotoxcicity CTL, NK and NKT
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?
Lord et al. Immunol Rev. 2003 Jun19331-8.
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Intracellular substrates for granzyme B

Also Caspase 10 Caspase 7 ICAD/DFF40
Lord et al. Immunol Rev. 2003 Jun19331-8.
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p53
P53 is induced by genotoxic stressors MDM-2
binds to p53 inactivation and degradation.
L11,L5
p53
MDM-2
Stabilize
Puma Noxa Bax PIDD Scotin Perp Fas
p53R2 GADD45 P25 14-3-3
ARF
Puma Strong apoptosis inducer has a BH3 domain
and a p53 binding site. Bind to Bax, Bak and
Bcl-2. No function in Bax KO fibroblasts.
Myc
Bcl-xL
DNA repair
Apoptosis
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UV and Radiation-induced Apoptosis
Few seconds
Cyto. C Oligo Bax Active Caspase 3,9
UV
Hela
Apoptosis
4 hr
Mitochondria (2 hr) normal cytosol or Mcl-1
inhibit cyto C release UV degradates Mcl-1
Bax
Bim
Bax/Bcl-xL
Cyto. C
Mcl-1-/- mice are sensitive to radiation.
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Autophagy and Autophagic Cell Death
Autophagy in Greek is to eat oneself. It is
the cells major regulated mechanism for
degrading long-lived proteins and organelles,
especially under nutrient or growth factor
deprivation conditions, cellular architectural
remodeling during developmental transitions, or
remove damaged cytoplasmic components during
oxidative stress, infection, and accumulation of
protein aggregates. It occurs at low basal levels
in all cells for cytoplasmic and organelle
turnover. For Death or for Survival?
Nature Reviews Molecular Cell Biology 6, 79-87
(2005)
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Autophagic Cell Death
Furuya et al. Autophagy 2004

• A form of programmed cell death (type II).
• Due to excessive cellular autophagy.
• Early degradation of organelles but preservation
  of cytoskeletal elements until late stages.
• Caspase activation and DNA fragmentation occur
  very late (if at all).
• No tissue inflammatory response.
• Occurs primarily during development or
  homeostatic organ involution and in tumor cells
  treated with chemotherapeutic agents.
• Possess morphologic features of apoptotic and
  necrotic cell death. Does autophagy causes
  apoptosis or necrosis?
• There is no demonstration of cell death
  resistance in apoptotic-competent cells lacking
  autophagy genes.

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• Story Completed?
• Apaf-1-/-, embryonic lethal. Liver cells can
  still generate normal lymphocytes
• Caspase-9-/- or Apaf-1-/- still has caspase 3.7
  activation.
• Axon degeneration? Caspases, Bax and Bak are not
  involved and Bcl-2 can not block.
• Many apoptosis molecules are not connected.
• 5. Relationship to autophagy

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Terms

• DED death effector domain
• CARD caspase recruitment domain
• CAD caspase-activated deoxyribonuclease
• ICAD CAD inhibitor
• FADD Fas-associated death domain
• FLICE Fas-like interleukin-1? converting enzyme
• DISC death inducing signaling complex
• AIF apoptosis inducing factor
• Apaf-1 apoptotic protease-activating factor-1
• Apoptosome Cyt.C, Apaf-1, dATP/ATP and
  pro-caspase 9
• Smac second mitochondria-derived activator of
  caspase.
• DIABLO direct IAP binding protein with low pI